You should definitely be using strict and warnings, for starters.
Next, do you really intend to update
$hash{$Query}[3] = $one;
$hash{$Query}[4] = $two;
in all 4 blocks? That seems strange.
If you didn't intend that, what I'd suggest is modifying your code to simplify things - it would also make debugging simpler.
while (<FH>) {
my ($Query, $Score, $Start, $End, $one, $two) = split;
my $aref=[$Score, $Start, $End, $one, $two];
if ( ! exists ( $hash{$Query} ) ) {
$hash{$Query} = $aref;
}
else {
if ( $hash{$Query}[1] > $Start ) {
$hash{$Query} = $aref;
}
}
if ( ! exists ( $hash2{$Query} ) ) {
$hash2{$Query}= $aref;
}
else {
if ( $hash2{$Query}[2] < $End ) {
$hash2{$Query} = $aref;
}
}
}
That might make things a bit easier to debug...
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